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HIV reverse transcriptase:

the molecular gymnast

When HIV or other retroviruses infect a cell, their goal is to store their genetic information into the DNA in the nucleus. However, there is a problem. Within the virion, genetic information is stored on single strands of RNA. After the virion fuses with a target cell, this RNA must be copied into a complimentary DNA, then the RNA must be digested and removed, and finally a second strand of DNA must be synthesized which is complimentary to the first strand. This series of reactions, shown in a simplified form in the figure to the left, results in a double stranded DNA molecule which now encodes the virus genome. This DNA is then imported to the nucleus and inserted in the host cell's chromosome.

Remarkably, one enzyme catalyzes all the reactions required for this transformation. That enzyme is reverse transcriptase (RT). Not surprisingly, this enzyme is a major target for anti-retroviral drugs. In our research, we are picking apart the complex mechanisms through which this enzyme functions using single molecule techniques. We have observed that RT can flip between opposite orientations on nucleic acids, allowing the enzymatic activity to switch between DNA polymerization and RNA cleavage. We have also seen that the enzyme can shuttle between distal ends of long duplexes of nucleic acids.

Flipping and shuttling allow RT to perform several key tasks in the HIV infection pathway, and it is therefore important to understand the molecular mechanism of these delicate motions. We also are curious how different antiretroviral drugs bias the motion of HIV and push it towards different enzymatic activities. Using state of the art biophysical techniques, we are probing how RT switches between different orientational states and recognizes uniques nucleic acid substrates.

DNA-binding proteins from starved cells:

crystallizing the genome

Life must often survive in harsh environments, which can cause damage at every scale from the level of organs to individual strands of DNA. This is especially true for single celled organisms such as bacteria, which are far too small to control their local temperature or store nutrients for when food runs out. Bacteria have therefore evolved a defensive state, called stationary phase, which allows them to wait out periods of stress such as starvation or temperature that would kill other cells. During stationary phase, the chromosome of the bacteria undergoes a remarkable transformation to a crystalline state (pictured to the right).

The enzyme responsible for this transformation is called Dps (DNA-binding proteins from starved cells).The mechanism by which Dps protects DNA in this state remains a mystery. Previously, Dps-DNA structures have only been observed in fixed cells, hampering efforts to investigate the dynamic properties of this phenomena. We are therefore developing tools to visualize Dps-DNA structures in live cells using fluorescence. Working with the Meyer Lab at TU Delft, we hope to use the tools to unravel the kinetics of the assembly of Dps on DNA and discover the mechanism of DNA protection. Cells exposed to antibiotics frequently use stationary phase to survive the damaging effects of the drugs, so if we can fully understand this process we may find a way to boost the potency of existing antibiotics by disabling the activity of Dps.